GP64-null baculoviruses pseudotyped with heterologous envelope proteins for gene therapy

ABSTRACT

A pseudotyped baculovirus comprises a deletion, inactivation or reduction from regulation of a baculovirus envelope protein gene, and is engineered to express an envelope protein from another virus or cell, or another protein or molecule that facilitates entry of the baculovirus into a non-host cell, or provided with a heterologous envelope protein or another protein or molecule that facilitates entry of the baculovirus into a non-host cell by other suitable means. Such baculoviruses can be used to efficiently deliver genes to mammalian cells or organisms, and such genes can be expressed either from the baculovirus genome, or integrated into the mammalian cell genome, and can be used for expression of proteins such that purification of secreted or other protein products does not require removal of contaminating baculovirus particles or baculovirus envelope proteins.

REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation application of copending application Ser.No. 09/925,365, filed Aug. 9, 2001, entitled “GP64-NULL BACULOVIRUSESPSEUDOTYPED WITH HETEROLOGOUS ENVELOPE PROTEINS”, which claims aninvention which was disclosed in Provisional Application No. 60/224,612,filed Aug. 11, 2000, entitled “A GP64-NULL BACULOVIRUS PSEUDOTYPED WITHTHE VESICULAR STOMATITIS VIRUS G PROTEIN THAT IS INFECTIOUS ANDPROPAGATES IN Sf9 CELLS”. The benefit under 35 USC § 119(e) of the U.S.provisional application is hereby claimed, and the aforementionedapplications are hereby incorporated herein by reference in theirentireties.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with Government support under Grant No.AI33657, awarded by the NIH. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The invention pertains to the field of virology. Moreparticularly, the invention pertains to a genetically engineeredbaculovirus in which the native envelope protein is absent, and thevirus particles are pseudotyped with an envelope protein from anothervirus..

[0005] 2. Description of Related Art

[0006] Baculoviruses constitute a family of viruses that are pathogenicTo certain insect species, but do not appear to productively infectother invertebrates or vertebrates. Baculoviruses, such as theAutographa californica Multicapsid Nucleopolyhedrovirus (AcMNPV), havebeen developed as biological control agents and as protein expressionvectors. AcMNPV also serves as the primary model system for studies ofbaculovirus gene regulation and structure. Baculoviruses have also beenused to deliver genes into mammalian cells, and can be used as vectorsfor human gene therapy.

[0007] AcMNPV has a large double stranded DNA genome (134 kbp) andproduces two virion phenotypes during the infection cycle. One virionphenotype, the Occlusion Derived Virus or ODV, is adapted for survivalin the environment and propagation of infection from animal to animal,through oral transmission and infection of the midgut epithelial cells.In contrast, the other virion phenotype (Budded Virus or BV) is adaptedfor propagation of infection from cell to cell throughout the animal,after infection is established in the midgut. BV efficiently enter manycell types in the infected animal, including most notably: hemocytes,tracheal epithelial cells, and fatbody cells. The infection cycle iscompleted when ODV are assembled (enveloped) and occluded withinocclusion bodies in the nuclei of these secondarily infected cells.Occlusion bodies are then released by cell lysis.

[0008] Because BV are generated only after successful infection of themidgut epithelial cells, BV appear to have adopted a strategy ofpromiscuous infection of many insect cell types. Studies of baculovirusBV entry into mammalian cell lines and cultured primary cells show thatin culture, BV from AcMNPV can efficiently enter primary rat hepatocytesas well as a number of human cell lines, although baculoviruses do notproductively replicate there. When a reporter gene driven by a mammalianpromoter is inserted into the AcMNPV genome, expression can be readilydetected in many mammalian cell types. In contrast, gene expressioncould not be detected from a reporter under the control of thebaculovirus polyhedrin promoter. Thus, baculoviruses appear toefficiently enter mammalian cells and selectively express genes underthe control of mammalian promoters. Thus, baculovirus BV can be aneffective means for gene delivery to mammalian cells, as gene therapyagents.

[0009] Indeed, several features of baculoviruses are highly desirablefor the development of baculoviruses as potential vectors for genetherapy. These include the capacity of the baculovirus genome toaccommodate very large insertions of foreign DNA, the inability of thevirus to replicate within mammalian cells, and the apparent absence ofexpression of most baculovirus genes. Other studies have shown thatbaculoviruses incorporating selectable markers (such as the neomycinphosphotransferase II gene) under a mammalian regulatory context, can beused to generate stably transformed mammalian cell lines.

[0010] The AcMNPV GP64 protein is an essential virion protein that isinvolved in both receptor binding and membrane fusion during viralentry. GP64 is also required for viral assembly and efficient productionof budded virions (BV) during viral exit.

[0011] During virion entry, the AcMNPV GP64 protein is involved inbinding of virions to host cells. GP64 also mediates low pH triggeredmembrane fusion during entry by endocytosis. Genetic studies withgp64-null viruses (containing a gp64 knockout) showed that GP64 is alsonecessary for efficient virion budding from the cell surface.Interestingly, AcMNPV viruses containing C-terminal truncations of GP64that removed portions or all of the cytoplasmic tail domain (CTD) didnot show the same severity of the defect in budding as the complete GP64deletion. This indicates that the CTD is not required for efficientbudding and that some other feature of GP64 is important for virionassembly and budding. Although the absence of GP64 resulted in anapproximately 98% reduction in virion budding, deletion of the CTDresulted in only an approximately 50% reduction in budding efficiency,indicating that other portions of the GP64 protein may play importantroles in budding.

[0012] GP64 is highly conserved among a number of baculoviruses (such asAcMNPV and OpMNPV) that are relatively closely related, yet several moredistantly related baculoviruses possess an unrelated envelope proteinthat appears to serve as a functional homolog of GP64. The major BVenvelope proteins from two of these more distantly related viruses,SeMNPV (Se8) and LdMNPV (Ld130), are both envelope fusion proteins andthus serve at least one of the important functions of GP64. However,these proteins and a homolog from XcGV show a higher degree ofdivergence than that observed among GP64 proteins. It has therefore beenproposed that GP64 may represent a more recent acquisition of anenvelope glycoprotein in the Baculoviridae. Several orthomyxovirusescontain an envelope protein, GP75, that is phylogenetically related tothe baculovirus GP64 protein. The GP75 proteins have been identifiedfrom only a small subset of the orthomyxoviruses and GP75 is distinctfrom the HA proteins found in other orthomyxoviruses. Therefore, it ispossible that the GP75 protein was also recently acquired by a member ofthe orthomyxovirus family.

[0013] In a previous study (Barsoum et al., Hum Gene Ther. 8: 2011-8(1997), the complete disclosure of which is hereby incorporated hereinby reference), a baculovirus expressing the VSV-G protein was reportedto have an enhanced ability to transduce mammalian cells. In that study,G was expressed in the presence of wt GP64 , presumably generatingvirions with a mosaic of GP64 and G protein in the envelopes. G proteindid not appear to interfere with the infectivity of the virus in insectcells but enhanced infectivity of mammalian cells. However, VSV-Gprotein expression in a baculovirus expression vector in the presence ofwt GP64 resulted in a report of virions that were distended andsometimes contained tail-like projections. A potential problem with theutilization of baculovirus virions (BV) containing GP64 for mammaliantransduction in vivo, is the rapid detection of GP64 and inactivation ofthe virus. The use of VSV-G protein or other suitable envelope ormembrane proteins substituted for GP64 in the BV envelope couldtherefore provide benefits for use of baculoviruses in vivo.Retroviruses pseudotyped with VSV-G appear to be more resistant toinactivation by complement than wild type retroviruses, and this mayalso be true for G-pseudotyped baculovirus particles.

[0014] In other enveloped viruses, the role of the major envelopeprotein in virion budding is highly variable. For example, retrovirusessuch as HIV-1 or RSV do not require the envelope protein (Env) forvirion budding, although virions generated in the absence of Env are notinfectious. In contrast, envelope proteins from influenza viruses arebelieved to encode important functions necessary for virion budding andalso influence virion morphology, and these important functions arethought to be redundant in the hemagglutinin (HA) and neuraminidase (NA)proteins of Influenza A virus. Rhabdoviruses such as VSV and Rabiesviruses require the major envelope protein (G protein) for efficientbudding. In the absence of G, budding of VSV or Rabies virions isreduced by approximately 97%. Heterologous proteins substituted for Gcan partially complement virion budding in VSV and Rabies rhabdoviruses,and studies suggest that important signals necessary for efficientbudding reside in non-specific signals in the cytoplasmic tail domain.Efficient budding of VSV in the absence of intact G protein can bereconstituted by providing only a ‘stem’ region containing the membraneproximal portion of the G protein ectodomain (12 amino acids) combinedwith the transmembrane and cytoplasmic tail domains. The small ‘stem’region appears to be a functional budding domain necessary to promoteefficient budding of VSV in the absence of the majority of the Gprotein.

[0015] One hypothesis to explain the synergistic roles of variousproteins in the budding process is the push-pull model, in which thepush represents the role of matrix and perhaps other proteins on theinner surface of the plasma membrane, and the pull represents the roleof the membrane proteins within and on the exterior of the membrane.Budding may be accomplished by the concerted or synergistic effects ofthe two components. While a very low level of budding may be observed inthe absence of one component, efficient budding would require theactivities of both components.

[0016] In certain retrovirus and rhabdovirus systems, heterologousenvelope proteins can complement the absence of the endogenous envelopeprotein. Virions carrying a heterologous envelope protein are referredto as ‘pseudotyped’ viruses. Pseudotyped virions have been used forapplications such as gene therapy, but also serve as valuable tools fordissecting the functions necessary for assembly of mature virions andbudding at the cell surface. Thus, to better understand the requirementsfor baculovirus budding, we investigated whether heterologous viralglycoproteins can complement the deletion of the gp64 gene from theAcMNPV genome.

SUMMARY OF THE INVENTION

[0017] We investigated whether a heterologous viral envelope protein,the Vesicular Stomatitis Virus (VSV) G protein, can complement thedeletion of GP64 in a gp64-null baculovirus, vAc⁶⁴⁻. To address thisquestion, we generated a stably transfected insect Sf9 cell line(Sf9^(VSV-G)) that inducibly expresses the VSV-G protein upon infectionwith AcMNPV. Sf9^(VSV-G) and Sf9 cells were infected with vAc⁶⁴⁻ andcells were monitored for infection and for movement of infection fromcell to cell. vAc⁶⁴⁻ formed plaques on Sf9^(VSV-G) cells, but not on Sf9cells. Plaques formed on Sf9^(VSV-G) cells were observed only afterprolonged intervals. Passage and amplification of vAc⁶⁴⁻ on Sf9^(VSV-G)cells resulted in pseudotyped virus particles that contained theVSV-G-protein. Cell-to-cell propagation of vAc⁶⁴⁻ in the G-expressingcells is delayed in comparison to wt AcMNPV, and growth curves showedthat pseudotyped vAc⁶⁴⁻ are generated at titres of approximately 10⁶ to10⁷ infectious units (IU)/ml, compared with titres of approximately 10⁸IU/ml for wt AcMNPV in the same cells.

[0018] Propagation and amplification of pseudotyped vAc⁶⁴⁻ virions inSf9^(VSV-G) cells indicates that the VSV-G-protein possesses thenecessary signals for baculovirus BV assembly and budding at the cellsurface, or otherwise facilitates production of infectious baculovirusvirions. The functional complementation of gp64-null viruses by VSV-Gprotein is further demonstrated by identification of a vAc⁶⁴⁻-derivedvirus that acquired the G gene through recombination with Sf9^(VSV-G)cellular DNA. Gp64-null viruses expressing the VSV-G gene are capable ofproductive infection, replication, and propagation in Sf9 cells. We thusdemonstrate herein that several functions of GP64 can be substituted bythe VSV-G protein, and we provide the first example of functionallypseudotyped baculovirus virions. We further demonstrate that the VSV-Gand two other heterologous envelope protein genes (i.e., two different Fproteins from baculovirus Group 2 NPVs) can be engineered into thegp64-null virus genome and functionally complement the absence of GP64.Our results are consistent with a push-pull model for baculovirusbudding.

[0019] The present invention shows that by expressing VSV-G or anotherheterologous envelope protein (whether from a cell line, or from thevirus), the virus is able to propagate and to infect insect cells. Thus,the VSV-G protein and other heterologous envelope proteins can beinserted into baculovirus virions that contain no GP64 (i.e., agp64-null baculovirus). We propose that this system is useful forgenerating baculoviruses targeted to specific cell types (depending onthe type of protein or other molecule used to replace GP64). Also, GP64is a target of immune recognition by the mammalian host (in vivo), andremoving GP64 allows the engineered viruses to escape immunesurveillance and subsequent inactivation. Immune recognition of GP64 hasbeen demonstrated with recombinant baculoviruses in vivo (injected intomice).

[0020] In addition, gp64-null viruses can be used to great advantage inprotein expression systems. A foreign gene for expression can be clonedinto the GP64-null virus and the virus propagated in a complementingcell line. When the virus is used to infect normal host cells, theforeign protein is expressed, but no virus particles are budded into theculture medium. Thus, purification of expressed foreign proteins isfacilitated, as contaminating virus particles do not need to be removed.

[0021] We expressed VSV-G (and two other heterologous viral envelopeproteins) in the absence of GP64 and found that VSV-G complements virioninfectivity and possibly virion budding, although the efficiency ofinfectious virion production appears to be relatively low. Thisrepresents the first example of pseudotyping of baculovirus virions inthe absence of the baculovirus GP64 protein. We propose that suchpseudotyped baculovirus virions are useful in potential gene therapyapplications.

BRIEF DESCRIPTION OF THE DRAWING

[0022]FIG. 1 shows a graphic map of the plasmid pSM8141-VSV-G.

[0023]FIG. 2 depicts the procedure used for vAc⁶⁴⁻ amplification inSf9^(VSV-G) cells.

[0024]FIG. 3 shows data from one step growth curves.

[0025]FIG. 4 shows a graphic map of the primer pairs used in PCRanalysis of the p10 locus of AcMNPV in wt, vAc⁶⁴⁻, and Sf9^(VSV-G) virusisolates.

DETAILED DESCRIPTION OF THE INVENTION

[0026] In a previous study, it was shown that when the vesicularstomatitis (VSV) G-protein was expressed from a recombinant AcMNPVbaculovirus, the presence of G-protein in BV appeared to enhanceinfectivity in mammalian cells. In that study, BV presumably containedboth VSV-G protein and GP64. In the current study, we investigatedwhether VSV-G protein alone was capable of complementing both virionbudding and infectivity in the context of a gp64-null virus, vAc⁶⁴⁻. Toexamine this question, we first generated and characterized aSf9-derived cell line that inducibly expresses the VSV-G protein uponinfection with AcMNPV. The cell line, Sf9^(VSV-G). was then infectedwith vAc⁶⁴⁻ and cells were monitored for movement of infection from cellto cell. Using this procedure, we generated pseudotyped virions thatcontain the VSV-G protein and were able to propagate infection from cellto cell in G-expressing cells, but not in Sf9 cells that do not expressVSV-G. Although cell-to-cell propagation of the gp64-null virus wasdelayed in comparison to wt AcMNPV propagation in the G-expressingcells, growth curves showed that pseudotyped virions were generated attitres of approximately 10⁶ to 10⁷ IU/ml, compared with titres ofapproximately 10⁸ for wt AcMNPV in the same cells.

[0027] We demonstrate that the G protein can complement a deletion ofGP64. Expression of G from a cell line is sufficient to complement theproduction of infectious virus particles that could be passaged inG-expressing cells. Although the levels of virions generated aresubstantially lower than from wt AcMNPV, the success of complementationis underscored by the observation that homologous recombination betweenthe null virus (vAc⁶⁴⁻) and the Sf9^(VSV-G) cells results in virusesthat express the VSV-G protein and are able to propagate infection inSf9 cells. We have also confirmed this result by directly cloning theVSV-G protein gene into the polyhedrin locus of a gp64-null AcMNPVvirus. Similar results were obtained.

[0028] Construction of Plasmid pSM8141-VSV-G

[0029] Referring to FIG. 1, a graphic map of the plasmid pSM8141-VSV-Gis shown. Plasmid pSM8141-VSV-G contains a VSV-G gene under the controlof an AcMNPV polyhedrin (PH) promoter and a B-glucuronidase (GUS) geneunder the control of both PH and p10 promoters from AcMNPV. Each genecassette is terminated by an SV40 polyA cleavage and addition site. Thetwo genes are flanked by Left and Right Arm sequences from the AcMNPVp35/hr5 region and the me53 region, respectively. To construct theplasmid, the Vesicular Stomatitis Virus G gene, derived from the Indianaserotype of VSV, was isolated from plasmid VSVG-BP95NOTSV as a 1692 bpBam HI fragment containing the entire VSV-G open reading frame. The VSVG-protein gene sequence is found under Genbank accession no. NC 001560,which contains the entire VSV genome sequence; the G-protein genesequence is found at nucleotides 3049-4713. The VSV-G coding gene wasinserted into the unique Bam HI site of a dual expression p 10 locustransfer plasmid, between a polyhedrin promoter and an SV40 terminator,to create plasmid pSM8135. The presence and orientation of the VSV-Ggene was confirmed by sequencing using primers located in the polyhedrinpromoter. As a marker for expression in infected insect cells, a BglII-Bam HI fragment containing a polyhedrin promoter andBeta-glucuronidase (GUS) reporter gene was inserted into the Bgl II siteof plasmid pSM8135 to create plasmid pSM8141-VSV-G.

[0030] Generation of Cell Line Sf9^(VSV-G) and Propagation of vAc⁶⁴⁻ inSf9^(VSV-G) Cells

[0031] To generate cells expressing VSV-G protein, Sf9 cells adapted toserum free medium (ESF921) were plated in T75 flasks (7.5×10⁶ cells perflask) and flasks were transfected with 2 μg pSM8141 plus 1 μg pIE-neo,2 μg pSM8141 alone, or mock transfected (no DNA added). Aftertransfection, cells were incubated in ESF921 medium for 24 hours, andthen resuspended, diluted 1:4 and replated in T75 flasks. ESF921 mediumwas replaced with ESF921 containing 1 mg/ml G418. After two weeks, cellswere monitored to confirm that all mock transfected cells were dead.Small cell colonies that grew only from the cells transfected withpSM8141 plus pIE-neo were selected as single, well isolated colonies,and were picked using sterile micropipettor tips and transferred toindividual wells of a 24 well dish, and cultured in ESF921 plus 5% FBS.A cell line derived from one colony was selected and designatedSf9^(VSV-G).

[0032] Cell lines Sf9, Sf9^(Op1D), and Sf9^(VSV-G) were propagated at27° C. in TNMFH medium containing 10% fetal bovine serum. Sf9^(Op1D)cells were used only at passages under 250. The wild type (wt) AcMNPVvirus used was AcMNPV strain E2. Construction of the gp64-null virus,vAc64⁻, was described previously by Oomens et al. in “Requirement forGP64 to drive efficient budding of Autographa californica MulticapsidNucleopolyhedrovirus,” Virology 254: 297-314 (1999), the completedisclosure of which is hereby incorporated herein by reference.Infectious vAc64⁻ was generated in Sf9^(Op1D) cells by infecting cellsat a multiplicity of infection (MOI) of 1, followed by harvest of virusat approximately 3 days post infection. vAc64⁻ was titred on Sf9^(Op1D)cells. Virus stocks of vAc64⁻ were monitored for the presence of rescuedvirus containing the OpMNPV gp64 gene by infecting Sf9 cells at low MOI(approximately 1×10⁻² to 1×10⁻⁴), followed by prolonged incubation andobservation for infected cells capable of propagating infection.

[0033] To determine if the presence of VSV-G protein is sufficient tofacilitate the production of infectious baculovirus in the absence ofGP64, a gp64-null virus (vAc⁶⁴⁻) containing no gp64 gene was used toinfect Sf9^(VSV-G) cells. Infected cells were examined for the capacityto propagate the gp64-null virus infection. Three cell lines wereinfected with virus vAc⁶⁴⁻: Sf9^(Op1D), a Sf9 derived line expressingthe OpMNPV GP64 protein, that was previously shown to complementvAc⁶⁴⁻(28, 30); Sf9 , a line that does not support propagation ofvAc⁶⁴⁻; and Sf9^(VSV-G), a line that inducibly expresses VSV-G protein.The gp64-null virus used for these experiments was propagated inSf9^(Op1D) cells as described previously in Virology 254: 297-314(1999), the complete disclosure of which is hereby incorporated hereinby reference. The virus inoculum contained the OpMNPV GP64 protein inthe envelope, but no gp64 gene in the viral genome.

[0034] Each cell line was infected with vAc⁶⁴⁻ and plaque formation wasexamined over an extended time period. As expected, vAc⁶⁴⁻ infection ofSf9^(Op1D) cells resulted in abundant formation of plaques as OpMNPVGP64 is known to complement the AcMNPV gp64 deletion. In contrast,vAc⁶⁴⁻ infection of Sf9 cells resulted in an initial infection of singlecells, but the virus failed to propagate from cell to cell and did notform plaques. In vAc⁶⁴⁻ infected Sf9^(VSV-G) cells, we initiallyobserved single infected cells and plaques were not clearly visible at5-7 days p.i. However, upon further incubation, plaques were detected inSf9^(VSV-G) cells by 10 days p.i., and had expanded significantly by16-18 days p.i. These observations indicate that the VSV-G protein iscapable of complementing the defect in the gp64 null virus, vAc⁶⁴⁻,although virus propagation appears to be delayed. The formation ofplaques indicates that the defects in both virion entry and exit arecomplemented.

[0035] To further examine this question, and to confirm that the nullvirus can be propagated and amplified in Sf9^(VSV-G) cells, we performedthe following experiment. Sf9, Sf9^(Op1D), or Sf9^(VSV-G) cells wereinfected with vAc⁶⁴⁻ at an MOI of 2.2×10⁻⁵ (1 IU per 4.5×10⁴ cells) andcells were incubated at 27° C. until they were 90% confluent. The mediumand cells from each well were then transferred into successively largerwells and then to T-flasks. At each step, cells were transferred whenthey reached approximately 90% confluency. Passage of vAc⁶⁴⁻ inSf9^(Op1D) cells in this manner resulted in a rapid propagation ofinfection, such that cell growth was arrested and all cells wereinfected after the third passage. This was expected, since the GP64protein expressed by the Sf9^(Op1D) cells complements the absence ofGP64 in virus vAc⁶⁴⁻. Attempted passage of vAc⁶⁴⁻ in Sf9 cells in thismanner resulted in no spread of infection. Although vAc⁶⁴⁻ appeared topropagate slowly in Sf9^(VSV-G) cells, continued passage resulted inincreasing numbers of infected cells until most cells were infected atpassage six or seven. Supernatants were then harvested and viruses weretitred on Sf9^(Op1D) cells, which are sensitive indicators of infectionby the gp64-null virus. We measured 6.2×10⁹ IUfrom the vAc⁶⁴⁻ viruspassaged in Sf9^(VSV-G) cells in the manner described above. Thus thevAc⁶⁴⁻ virus was amplified approximately 3.9×10⁸ fold in Sf9^(VSBV-G)cells in this experiment. Herein we refer to the vAc⁶⁴⁻ that wasamplified in Sf9^(VSV-G) cells as pseudotyped virus or ^(G)vAc⁶⁴⁻.

[0036] Expression of VSV-G in Sf9^(VSV-G) Cells

[0037] Because it was previously reported that expression of VSV-G istoxic in some cell lines, we used a strategy in which expression ofVSV-G in insect Sf9 cells was dependent on infection with AcMNPV. Theplasmid pSM8141-VSV-G, containing the VSV-G gene under the control of anAcMNPV polyhedrin promoter (FIG. 1), was constructed and cotransfectedinto Sf9 cells with the plasmid pIE1-neo, which contains the E. colineomycin phosphotransferase II gene under the control of the AcMNPV IE1promoter. G418 was used to select and clone a cell line that wasdesignated Sf9^(VSV-G) . To determine whether the VSV-G gene wasinserted into the cell line, and whether expression of VSV-G wasinducible by infection with AcMNPV, we examined induced (infected) anduninduced (mock-infected) Sf9^(VSV-G) cells by western blot analysis andimmunofluorescence microscopy, and compared VSV-G expression inmock-infected and infected Sf9^(VSV-G) cells.

[0038] VSV-G protein was detected in Sf9^(VSV-G) cells infected witheither wt AcMNPV or vAc⁶⁴⁻, but was not detected in mock-infected cells.An antiserum directed against the major capsid protein (VP39) was usedas an internal control to confirm infection. Detection of VSV-G byimmunofluorescence microscopy showed that VSV-G was detected from AcMNPVor vAc⁶⁴⁻ infected Sf9^(VSV-G) cells, but not from mock-infectedSf9^(VSV-G) cells or Sf9 cells infected with wt AcMNPV.Immunofluorescent staining of infected cells was consistent with VSV-Gpresence at the periphery of infected cells, indicating that G waslikely transported to the surface of these cells. Thus, infection ofcell line Sf9^(VSV-G) with wt AcMNPV or vAc⁶⁴⁻ results in the inductionof VSV-G protein expression and G appears to be appropriately localizedwithin these cells.

[0039] SDS-PAGE and Western Blot Analysis

[0040] For western blot analysis of GP64, VSV-G, and VP39 proteins,cells were infected at an MOI of 1 and harvested at 46-75 hourspost-infection (p.i.), then examined for VSV-G protein expression andVP39 protein expression, using monoclonal antibodies (anti-GP64 MAbAcV5, anti-VSV-G MAb P5D4, or anti-VP39 MAb P10). Samples were preparedfor western blot analysis in the following manner. Cell extracts frominfected or uninfected cells were lysed in 1× Laemmli buffer (125 mMTris, 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, 0.001% bromophenolblue, pH 6.8) and heated to 100° C. for 5 minutes prior to storage at−20° C. or electrophoresis. Virions of wt AcMNPV or pseudotyped^(G)vAc64⁻BV were prepared from tissue culture supernatant bycentrifugation at 80,000×g for 75 minutes at 4° C. through a 25% sucrosecushion in PBS, and subsequent resuspension of the pellet in 1× Laemmlibuffer. Samples were heated to 100° C. for 5 minutes and electrophoresedthrough 10% SDS-PAGE gels. Approximately 2.6×10⁴ cells or 8×10⁶ virionswere electrophoresed in each lane. Gels were blotted onto Immobilon-Pfilters and incubated with the following primary monoclonal antibodies:AcV5, an anti-GP64 MAb; MAb P10, an anti-VP39 MAb; or P5D4, an antiVSV-G MAb. Monoclonal antibodies were diluted 1:100, 1:1,000, and1:100,000, respectively in TBST (10 mM Tris pH 8, 150 mM NaCl, 0.05%Tween 20) with 0.02% sodium azide. After washing, blots were incubatedwith a secondary antibody consisting of a goat-anti-mouse IgG-alkalinephosphatase conjugate at a dilution of 1:10,000. Western blots were thenprocessed as described previously by Blissard, et al. in “BaculovirusGP64 envelope glycoprotein is sufficient to mediate pH dependentmembrane fusion,” J. Virol. 66: 6829-6835 (1992), the completedisclosure of which is hereby incorporated herein by reference.

[0041] To confirm that the amplified virus (^(G)vAc⁶⁴⁻) did not resultfrom contamination with wt AcMNPV or a gp64-null virus that acquired theOpMNPV gp64 gene during prior propagation in Sf9^(Op1D) cells, we usedwestern blot analysis to examine cells infected with either wt AcMNPV,vAc⁶⁴⁻, or ^(G)vAc⁶⁴⁻. The GP64 protein was detected from cells infectedwith wt AcMNPV and from virus infections in Sf9^(Op1D) cells, whichconstitutively expresses OpMNPV GP64. In addition, a weak GP64 signalwas frequently observed from cells infected with vAc⁶⁴⁻. Because vAc⁶⁴⁻was previously passaged in Sf9^(Op1D) cells and carries wt OpMNPV GP64in the envelope, low levels of GP64 detected from these samples canresult from GP64 carried in with the inoculum virus. However, the GP64protein was not detected from Sf9 or Sf9^(VSV-G) cells infected with^(G)vAc⁶⁴⁻. VSV-G was detected in all infected Sf9^(VSV-G) cells, asexpected.

[0042] Interestingly, a strong VSV-G signal was detected in extractsfrom all cells infected with the pseudotyped virus, ^(G)vAc⁶⁴⁻. Aplausible explanation for this result was that some of the ^(G)vAc⁶⁴⁻virus may have acquired the VSV-G gene during passage through theSf9^(VSV-G) cells and thus was expressing G protein from the virusgenome. This possibility was addressed in detail in subsequentexperiments. In the current experiments, we found that GP64 was notdetected in Sf9 or Sf9^(VSV-G) cells infected with the G-pseudotypedvirus (i.e., ^(G)vAc⁶⁴⁻). Thus, the observed propagation of vAc⁶⁴⁻ inSf9^(VSV-G) cells was not due to contamination with a virus expressingGP64. In addition, these data confirm that the gp64-null virus (vAc⁶⁴⁻)can be propagated in G-expressing cells, in the absence of GP64.

[0043] Immunofluorescence Microscopy

[0044] For immunofluorescent detection of VSV-G protein in Sf9 orSf9^(VSV-G) cells infected with wt AcMNPV or vAc⁶⁴⁻, cells were infectedwith the indicated viruses at an MOI of 10, then fixed at 40 hours p.i.and immunostained with MAb P5D4 and goat-anti-mouse FITC. Cells wereexamined and photographed by epifluorescence microscopy. Forimmunofluorescence staining, 1×10⁵ Sf9 or Sf9^(VSV-G) cells were platedper well on 2-well slides and cells were allowed to attach for 1 hour,then mock-infected or infected with AcMNPV or vAc⁶⁴⁻ (MOI 10) for 1hour. At 40 hours p.i., cells were washed 3 times with 1 ml PBS (pH 6.4)per well, then fixed in 100% methanol at −20° C. for 10 min. Cells werethen air-dried 10 minutes and rehydrated in 300 μl buffer A (5% filteredFBS, 0.1% saponin, 1X PBS) for 10 min. Cells were then incubated withanti-VSV-G antibody (P5D4, mouse ascites fluid, Sigma), diluted 1:10,000in buffer A (described above) for 45 minutes at room temperature (RT).After 3 washes with 300 μl Buffer A (10 min/wash), cells were incubatedwith a 1:100 dilution of goat anti-mouse IgG FITC conjugate for 30minutes at RT. Cells were washed 4× with buffer A, then sealed inGelMount and viewed on an Olympus IX70 epifluorescence microscope.

[0045] Plaque Assays, Growth Curves and TCID₅₀ Assays

[0046] Plaque assays were performed in six well plates, as previouslydescribed in J. Virol. 66: 6829-6835 (1992), the complete disclosure ofwhich is hereby incorporated herein by reference. Sf9, Sf9^(Op1D) andSf9^(VSV-G) cells were plated at 1.5×10⁶ cells/well, and after a 1 hourattachment period, a monolayer of cells (Sf9^(Op1D), Sf9^(VSV-G), orSf9) was infected with vAc⁶⁴⁻ at several dilutions. Cells were monitoredfor infection and plaque formation over an 18 day period, and at 10 or18 days, each well was overlaid with 50 μg/ml neutral red in 1% agar andinfected cells were examined at the indicated intervals (5, 7, 10, 14,or 16 days). Infected cells containing occlusion bodies appear as darkcells against the background of lighter cells. Plaque formation andmorphology in vAc ⁶⁴⁻ infected Sf9^(Op1D), Sf9^(VSV-G), and Sf9 cellmonolayers were examined after 10 and 18 days, respectively. Growthcurves were carried out by a modification of the protocol described inVirology 254: 297-314, the complete disclosure of which is herebyincorporated herein by reference. Sf9 cells were infected with AcMNPV,and Sf9^(Op1D) and Sf9^(VSV-G) cells were infected with vAc64⁻ at an MOIof 5. After an initial 1 hour infection period, cells were washed 3×with TNMFH and supernatants were collected at the indicated time points.Data from each time point represents accumulated infectivity frominfection through the indicated time. All supernatants were titred byTCID₅₀ assay on Sf9^(Op1D) cells.

[0047] In our initial analysis of vAc⁶⁴⁻ virus propagation inSf9^(VSV-G) cells, we observed that plaque formation was significantlydelayed, when compared with plaque formation by the same virus inSf9^(Op1D) cells. This could result from a delay in the infection cycle,low virus yield, lowered infectivity of the pseudotyped virus, or somecombination of these factors. To examine the kinetics of virionproduction in vAc⁶⁴⁻ infected Sf9^(VSV-G) cells, we generated a one stepgrowth curve of infectious virus production, and compared that curve tosimilar curves generated from wt AcMNPV infected Sf9 cells, and vAc⁶⁴⁻infected Sf9^(Op1D) cells. Because the infectivity of virions carryingVSV-G protein may differ from those carrying GP64, and because theobserved propagation of viruses in G-expressing cells was delayed, allvirus samples collected from growth curve experiments were titred onSf9^(Op1D) cells.

[0048]FIG. 3 shows growth curve data for the gp64-null virus (i.e.,vAc⁶⁴⁻) in cells expressing VSV-G (Sf9^(VSV-G) ) or OpMNPV GP64(Sf9^(Op1D)). For comparison, a growth curve of wt AcMNPV infected Sf9cells is also plotted. Cells were infected and supernatants collected atthe indicated times post infection, and virus yields were determined bytitration on Sf9^(Op1D) cells. Each virus-cell combination is indicatedin the inset. Each data point represents 3 individual infections, anderror bars are indicated. Each cell line (Sf9, Sf9^(VSV-G) orSf9^(Op1D)) was infected at an MOI of 5 with either wt AcMNPV or vAc⁶⁴⁻,and supernatants were harvested at the indicated times post infection.Supernatants were then titred on Sf9^(Op1D) cells. The temporal kineticsof the growth curves of all viruses were similar (FIG. 3), although peakvirion production of the G pseudotyped vAc⁶⁴⁻ appeared to lag behindthat of wt AcMNPV infected Sf9 cells and vAc⁶⁴⁻ infected Sf9^(Op1D)cells. For the two control infections (AcMNPV in Sf9 cells or vAc⁶⁴⁻ inSf9^(OP1D) cells), titres of 1×10⁷ IU/ml were observed by 24-48 hoursp.i. In contrast, vAc⁶⁴⁻ infected Sf9^(VSV-G) cells produced titres inthe range of 10⁵ IU/ml at 24 hours and approximately 10⁶ IU/ml by 48hours. Titres of the pseudotyped virus gradually increased to 10⁷ IU/mlat 168 hours p.i. AcMNPV infected Sf9 cells and vAc⁶⁴⁻ infectedSf9^(Op1D) cells generated titres of approximately 10⁸ IU/ml by 72-120hours p.i. Thus, while the kinetics of virus production were generallysimilar, the production of virus particles that were pseudotyped withVSV-G protein lagged slightly behind that of wt AcMNPV, and final yieldsof infectious virus were reduced by at least one log and representedapproximately 10% of the final yield from wt AcMNPV.

[0049] PCR Analysis

[0050] For preliminary PCR analysis, oligonucleotide primers specific toregions within the VSV-G, gp64, p35, or vp39 open reading frames weredesigned. Primer pairs were composed of oligonucleotides with thefollowing nucleotide sequences: Primer pair VSV-G =5′-tccgatccttcactccatctg-3′ (SEQ ID NO: 1) and5′-tagctgagatccactggagag-3′; (SEQ ID NO: 2) Primer pair gp64 =5′-gttgttattggctacaagggc-3′ (SEQ ID NO: 3) and5′-tgagtagagcgtggcgttgagc-3′; (SEQ ID NO: 4) Primer pair p35 =5′-cagaattcatgtgtgtaatttttccggtag-3′ (SEQ ID NO: 5) and5′-aatgctctagattatttaattgtgtttaatattac-3′; (SEQ ID NO: 6) Primer pairvp39 = 5′-cgggatccaatggcgctagtgcccgtgggtatgg-3′ (SEQ ID NO: 7) and5′-cgggatccgcgacggctattcctccacctgcttc-3′. (SEQ ID NO: 8)

[0051] PCR amplification reactions contained 0.3 μM of each primer in apair, 0.3 mM dATP, dCTP, dGTP, dTTP, 50 mM KCl, 10 mM Tris-HCL pH 8.3,1.5 mM MgCl, 0.1% Triton X100, and 0.4 U Taq DNA Polymerase in a finalvolume of 20 μl. Reactions were subjected to 94° C. for 3 minutes,followed by 3 cycles at an annealing temperature of 52° C., then 27cycles at an annealing temperature of 53° C., where each cycle consistsof denaturation at 94° C. for 30 seconds, annealing at the prescribedtemperature (above) for 40 seconds, and extension at 72° C. for 1.5minutes. The final extension was held at 72° C. for 10 minutes. Productswere electrophoresed on 1% agarose gels and stained with ethidiumbromide.

[0052] To amplify portions of the p10 locus from the genomes of wt andpseudotyped viruses, we used primer pairs that would amplify: a)fragments from only the intact p10 locus (FIG. 4, primer pairs A, B, andC) or b) portions of the VSV-G and p10 locus if the VSV-G gene wereintegrated at the predicted site (FIG. 4, primer pairs D and E). Each 50μl reaction contained 0.3 μM of each primer, 0.3 mM dATP, dCTP, dGTP,dTTP, 10 mM KCl, 10 mM(NH₄)₂S0₄, 20 mM Tris-HCl pH 8.8, 2 mM MgSO₄, 0.1%Triton X100, and 0.6 U Vent DNA polymerase. Amplification reactions wereheld at 94° C. for 3 minutes, followed by 30 cycles of 94° C. for 45seconds, 53° C. for 45 seconds, and 72° C. for 5 minutes. Finally,reactions were held at 72° C. for 5 minutes. Primer pairs consisted ofoligonucleotides with the following nucleotide sequences: Primer pair A= 5′-tgcgtgttgaagccgggatttg-3′ (SEQ ID NO: 9) and5′-gtcccgacagctgggacgcct-3′; (SEQ ID NO: 10) Primer pair B =5′-cgaatggctgttaccggtgacg-3′ (SEQ ID NO: 11) and5′-ctcgctatacactcgcatggag-3′; (SEQ ID NO: 12) Primer pair C =5′-cgatgcatatgtatggcatacc-3′ (SEQ ID NO: 13) and5′-gagtttgggaacaagtttgaagg-3′; (SEQ ID NO: 14) Primer pair D =5′-tgcgtgttgaagccgggatttg-3′ (SEQ ID NO: 15) and5′-gtgaagagtatcagtgtgcatg-3′; (SEQ ID NO: 16) Primer pair E =5′-gtagaaggttggttcagtagttg-3′ (SEQ ID NO: 17) and5′-gagtttgggaacaagtttgaagg-3′. (SEQ ID NO: 18)

[0053] Referring now to FIG. 4, a graphic map of the primer pairs usedin PCR analysis of the p10 locus of AcMNPV in wt, vAc⁶⁴⁻, andSf9^(VSV-G) virus isolates is shown. For PCR analysis of viral DNAs fromcell lysates of Sf9 cells infected with ^(G)vAc⁶⁴⁻ gene, severalspecific primer pairs (not shown) were used to examine isolates for thepresence of the OpMNPV gp64 gene or the VSV-G gene. The arrows in FIG. 4show the locations of the primers on the wt AcMNPV and vAc⁶⁴⁻ genomes,and on the putative genome of ^(G)vAc⁶⁴⁻ isolates, in which VSV-G couldhave integrated. DNAs from infected Sf9 cell lysates were used astemplates for PCR analysis of p10 locus in vAc⁶⁴⁻ (primer pairs A, B,and C), or the putative p10 locus in which VSV-G is predicted tointegrate into the ^(G)vAc⁶⁴⁻ genome (primer pairs D, and E). Thesequences of the gene-specific primer pairs are indicated above.

[0054] Electron Microscopy

[0055] For transmission electron microscopy, virions were purified fromcell culture supernatants, then fixed, embedded, sectioned and stained.For each virus preparation (wt AcMNPV or ^(G)vAc⁶⁴), 33 ml of infectedcell culture supernatants (representing 1×10⁹ or 3×10⁹ IU, respectively)were pelleted by centrifugation at 80,000×g for 75 minutes at 4° C.through a 25% sucrose cushion. The resulting virus pellet was fixed in2.5% gluteraldehyde in 100 mM cacodylate buffer (pH 7.2), then postfixedin 1.5% osmium tetroxide overnight at 4° C. After fixing, cells weredehydrated through a graded series of EtOH washes and embedded inSpurr's embedding medium. Ultrathin sections were stained by incubationfor 5-30 minutes in 2% uranyl acetate in H₂O, washed 3× in dH₂O, thenstained for 5 minutes in Reynolds lead citrate, and washed 5× in dH₂O.Sections were examined at magnifications of 15,000× and 70,000× at 80 or100 kV on a Phillips 201 transmission electron microscope.

[0056] To determine if virions generated in the presence of the VSV-Gprotein, and in the absence of GP64, were altered in morphology, we usedtransmission electron microscopy to compare ^(G)vAc⁶⁴⁻ virions withthose from wt AcMNPV. An obvious initial difference between preparationsof wt AcMNPV and ^(G)vAc⁶⁴⁻, was the presence of numerous vesicles ofvarying sizes. Such vesicles may result from vesicle budding mediated byexpression of the G protein. Vesiculation has been previously reportedin mammalian cells expressing VSV-G protein. Infectious virus titreswere lower in the ^(G)vAc⁶⁴⁻ preparation and virus particles were lessnumerous compared with wt AcMNPV preparations. However, ^(G)vAc⁶⁴⁻virions were clearly visible. wt AcMNPV BV consist of envelopedrod-shaped nucleocapsids. The envelope is typically composed of anapparently loosely adhering (lipid bilayer) membrane with a thickenedregion in the membrane, near one end of the rod-shaped nucleocapsid.These characteristics were also typical of BV from the ^(G)vAc⁶⁴⁻preparation. We did not typically observe nucleocapsids within enlargedor distended envelopes and nucleocapsids within larger vesicles were notobserved. Thus, although ^(G)vAc⁶⁴⁻ virions were less abundant,^(G)vAc⁶⁴⁻ virions appeared to be similar in morphology to those from wtAcMNPV and they were not morphologically distinguishable.

[0057] Examination of virions generated from the G-pseudotyped gp64-nullvirus used in this study showed clearly that virions were similar inmorphology to wt AcMNPV virions. Nucleocapsids were not observed withinvesicles or within virions that appeared as oval-shaped particles withtail-like structures as reported earlier.

[0058] Biochemical and Genetic Analysis of ^(G)vAc⁶⁴⁻ BV

[0059] Virus particles pseudotyped with VSV-G protein were examinedbiochemically for the presence of the G protein. ^(G)vAc⁶⁴⁻ virions wereprepared from supernatants of vAc⁶⁴⁻ infected Sf9^(VSV-G) cells aftermultiple passages in Sf9^(VSV-G) cells, then examined by Western blotanalysis. As a comparison, wt AcMNPV virions were examined in parallel.Preparations of ^(G)vAc⁶⁴⁻ virions contained substantial quantities ofG, but GP64 was not detected. In wt AcMNPV preparations produced in Sf9cells, G protein was not detected. Detection of the major capsidprotein, VP39, was similar in both wt AcMNPV, and ^(G)vAc⁶⁴⁻preparations.

[0060] Because VSV-G was detected at relatively high levels in cellsinfected with ^(G)vAc⁶⁴⁻, and VSV-G was also abundant in virionpreparations, it was possible that the VSV-G gene was acquired by thevAc⁶⁴⁻ virus through homologous recombination. We therefore investigatedwhether the VSV-G gene could be identified in DNA from ^(G)vAc⁶⁴⁻virions prepared after passage in Sf9^(VSV-G) cells. DNA prepared fromvirions of ^(G)vAc⁶⁴⁻, vAc⁶⁴⁻ (passaged in Sf9^(Op1D) cells), or OpMNPVwere used with a series of oligonucleotide primers to amplify portionsof the VSV-G ORF, the OpMNPV gp64 ORF, and the AcMNPV p35 and vp39 ORFs.Primers specific for portions of the AcMNPV p35 and vp39 ORFs wereincluded as positive controls for AcMNPV genes, and the OpMNPV gp64 ORFspecific primers were included as a control to confirm that viruses didnot acquire the gp64 gene during propagation in Sf9^(Op1D) cells. Asexpected, p35 and vp39-specific primers amplified the expected PCRproducts (900 and 1044 bp, respectively) from ^(G)vAc⁶⁴⁻ and vAc⁶⁴⁻ DNAsbut not from OpMNPV DNA. The OpMNPV gp64 specific primers amplified theappropriate (1259 bp) fragment from only the OpMNPV DNA, indicating thatthe OpMNPV GP64 gene was not present in ^(G)vAc⁶⁴⁻ and vAc⁶⁴⁻ DNAs.Using VSV-G specific primers, a 673 bp fragment was amplified from^(G)vAc⁶⁴⁻ virion DNA, but not from vAc⁶⁴⁻ virion DNA. Thus, these datasuggest that ^(G)vAc⁶⁴⁻ virions acquired the VSV-G gene duringpropagation in the Sf9^(VSV-G) cells.

[0061] If the ^(G)vAc⁶⁴⁻ virus acquired the G gene, this virus should nolonger require the Sf9^(VSV-G) cells for propagation. To determinewhether acquisition of the G gene would permit the ^(G)vAc⁶⁴⁻ virus topropagate independently of the Sf9^(VSV-G) cell line, we infected Sf9cells with a ^(G)vAc⁶⁴⁻ virus preparation that was passaged inSf9^(VSV-G) cells. This resulted in a spreading infection and the viruswas passaged twice in Sf9 cells (5 days per passage), then severalisolates were generated by limiting dilutions in Sf9 cells. G-specificprimer pairs were used to examine DNA from infected cell lysates by PCR.We found that the VSV-G gene was present in five virus preparationsgenerated in this manner, indicating that each contained viruses with acopy of the VSV-G gene. Although these viruses were able to propagate inSf9 cells, each isolate was negative for the OpMNPV gp64 gene,indicating that virus propagation in Sf9 cells was not due tocontamination with a virus that acquired the OpMNPV gp64 gene fromSf9^(Op1D) cells.

[0062] Because the plasmid used to generate the Sf9^(VSV-G) cell linewas derived from a transfer vector plasmid that was originally designedfor cloning into the AcMNPV p10 locus, we reasoned that acquisition ofVSV-G by the vAc⁶⁴⁻ virus may occur by homologous recombination at thep10 locus. We therefore used a PCR strategy to examine the p10 locus ofviral isolates passaged first in Sf9^(VSV-G) cells, then in Sf9 cells.FIG. 4 shows the PCR strategy. As expected, the appropriate PCR productswere identified from the parental virus, vAc⁶⁴⁻, when primer pairsspecific for the wt AcMNPV p10 locus were used. In addition, no PCRproducts were detected from vAc⁶⁴⁻ template when primers specific forthe predicted VSV-G insertion were used.

[0063] Interestingly, DNAs from ^(G)vAc⁶⁴⁻ that was passaged firstthrough the Sf9^(VSV-G) cells, then through Sf9 cells, were positive forboth sets of primers. This indicates that genotypes containing a) a wtp10 locus and b) a VSV-G insertion in the p10 locus, were both presentin the preparation. Other similarly derived isolates also showed thesame result, indicating that these virus preparations likely containedmixtures of parental viruses (vAc⁶⁴⁻) and recombinant viruses in whichthe G gene was inserted at the p10 locus. We propose that, because theserecombinant viruses abundantly express G protein, they may serve ashelper viruses for the defective vAc⁶⁴⁻ viruses containing no GP64envelope protein.

[0064] In summary, we found that the VSV-G protein was sufficient tocomplement the defect in the vAc⁶⁴⁻ (gp64-null) virus when G wasprovided by the cell line Sf9^(VSV-G). In addition this complementationappears to result in sufficient selection pressure so that the G genebecomes integrated into the genome of vAc⁶⁴⁻. We detected integration ofthe VSV-G gene in the p10 locus of vAc⁶⁴⁻ in several viral isolates, andfound that these viruses were capable of independent propagation in SF9cells.

[0065] We expressed VSV-G in the absence of GP64 and found that VSV-Gcomplements virion infectivity and possibly virion budding, although theefficiency of infectious virion production appears to be relatively low.This represents the first example of pseudotyping of baculovirus virionsin the absence of the baculovirus GP64 protein. We propose that suchpseudotyped baculovirus virions are useful in potential gene therapyapplications.

[0066] Baculoviruses are subdivided into two groups. While the buddedvirions from one group (Group 1 NPVs) contain the GP64 protein, thosefrom other groups (Group 2 NPVs and GVs) contain an unrelated protein(F-protein) that serves a similar functional role. We have demonstratedthat at least 2 of the F-proteins from Group 2 NPVs also complementinfectivity in the context of a gp64-null genotype.

[0067] Envelope proteins from Rhabdoviridae other than VSV (such as, forexample, rabies virus) also are expected to complement infectivity ofthe gp64-null baculovirus, as described herein, as are envelope proteinsfrom other families of viruses, such as, for example, Orthomyxoviridae,Paramyxoviridae, Filoviridae, Retroviridae, as well as herpesviruses,poxviruses and hepadnaviruses.

[0068] Finally, chimeric envelope proteins that consist of parts of GP64and parts of other envelope proteins, or parts of two or more differentenvelope proteins, also are expected to complement infectivity of thegp64-null baculovirus, as described here.

[0069] Accordingly, it is to be understood that the embodiments of theinvention herein described are merely illustrative of the application ofthe principles of the invention. Reference herein to details of theillustrated embodiments is not intended to limit the scope of theclaims, which themselves recite those features regarded as essential tothe invention.

1 18 1 21 DNA Vesicular stomatitis virus 1 tccgatcctt cactccatct g 21 221 DNA Vesicular stomatitis virus 2 tagctgagat ccactggaga g 21 3 21 DNAAutographa californica nucleopolyhedrovirus 3 gttgttattg gctacaaggg c 214 22 DNA Autographa californica nucleopolyhedrovirus 4 tgagtagagcgtggcgttga gc 22 5 30 DNA Autographa californica nucleopolyhedrovirus 5cagaattcat gtgtgtaatt tttccggtag 30 6 35 DNA Autographa californicanucleopolyhedrovirus 6 aatgctctag attatttaat tgtgtttaat attac 35 7 34DNA Autographa californica nucleopolyhedrovirus 7 cgggatccaa tggcgctagtgcccgtgggt atgg 34 8 34 DNA Autographa californica nucleopolyhedrovirus8 cgggatccgc gacggctatt cctccacctg cttc 34 9 22 DNA Autographacalifornica nucleopolyhedrovirus 9 tgcgtgttga agccgggatt tg 22 10 21 DNAAutographa californica nucleopolyhedrovirus 10 gtcccgacag ctgggacgcc t21 11 22 DNA Autographa californica nucleopolyhedrovirus 11 cgaatggctgttaccggtga cg 22 12 22 DNA Autographa californica nucleopolyhedrovirus12 ctcgctatac actcgcatgg ag 22 13 22 DNA Autographa californicanucleopolyhedrovirus 13 cgatgcatat gtatggcata cc 22 14 23 DNA Autographacalifornica nucleopolyhedrovirus 14 gagtttggga acaagtttga agg 23 15 22DNA Autographa californica nucleopolyhedrovirus 15 tgcgtgttga agccgggatttg 22 16 22 DNA Vesicular stomatitis virus 16 gtgaagagta tcagtgtgca tg22 17 23 DNA Vesicular stomatitis virus 17 gtagaaggtt ggttcagtag ttg 2318 23 DNA Autographa californica nucleopolyhedrovirus 18 gagtttgggaacaagtttga agg 23

What is claimed is:
 1. A method of administering gene therapy ordelivering recombinant or foreign polynucleotides to mammalian cells ororganisms, comprising the steps of: a) creating a genetically engineeredbaculovirus having a deletion, inactivation or downregulation of anenvelope protein gene of a progenitor baculovirus, from which saidengineered baculovirus is derived, wherein said genetically engineeredvirus is supplied, in trans or cis, with a heterologous envelope proteinor a protein or other molecule that facilitates entry of said engineeredbaculovirus into a cell that is not normally a host of said progenitorbaculoviru; b) further modifying said engineered baculovirus to expressa gene therapy agent or recombinant or foreign polynucleotide; and c)delivering said modified baculovirus into said mammalian cell ororganism.
 2. The method of claim 1, wherein said genetically engineeredbaculovirus has a property of more efficient entry into a non-host cellthan said progenitor baculovirus, from which said engineered baculovirusis derived.
 3. The method of claim 1, wherein said geneticallyengineered baculovirus has a property of more specific targeting of saidengineered baculovirus to a specific cell type than said progenitorbaculovirus, from which said engineered baculovirus is derived.
 4. Themethod of claim 1, wherein said genetically engineered baculovirus has aproperty of more effective evasion of mammalian immune systemrecognition or inactivation than said progenitor baculovirus, from whichsaid engineered baculovirus is derived.
 5. The method of claim 1,wherein said genetically engineered baculovirus is engineered to expressan envelope protein from Vesicular Stomatitis Virus.
 6. The method ofclaim 5, wherein said envelope protein from Vesicular Stomatitis Virusis VSV-G protein.
 7. A method of protein expression, comprising thesteps of: a) creating a genetically engineered baculovirus having adeletion, inactivation or dowenregulation of an envelope protein gene ofa progenitor baculovirus, from which said engineered baculovirus isderived, wherein said genetically engineered virus is supplied, in transor cis, with a heterologous envelope protein or a protein or othermolecule that facilitates entry of said engineered baculovirus into acell that is not normally a host of said progenitor baculovirus; b)further modifying said engineered baculovirus to express a recombinantor foreign polynucleotide; and c) delivering said modified baculovirusinto a suitable cell for expression of said recombinant or foreignprotein by said engineered baculovirus.
 8. The genetically engineeredbaculovirus of claim 1, wherein said genetically engineered baculovirusinfects and replicates in at least one permissive insect cell host ofsaid progenitor baculovirus.
 9. The genetically engineered baculovirusof claim 8, wherein said progenitor baculovirus envelope protein is gp64or a homologous envelope glycoprotein.